>Dear Netters:
>
>Can anyone help with information about the best way of
>inhibiting protein synthesis in Arabidopsis roots? We want to
>determine whether the rapid induction (<60 min) of certain
>genes by nitrate is part of the primary response to nitrate -
>i.e. whether it is independent of protein synthesis. So we
>need something that is effective, rapid and without
>side-effects (ideally!). The use of any protein synthesis
>inhibitors is fraught with difficulties because of secondary
>effects, but has anyone got any experience of using
>cycloheximide, for example, on Arabidopsis?
>>Thanks in advance for any help or advice.
>>Brian Forde
>>brian.forde at bbsrc.ac.uk>Biochemistry and Physiology Dept.
>IACR-Rothamsted
>Harpenden
>Herts AL5 2JQ
>UK
Many thanks for the replies that I received to the above message,
which I reproduce below. Basically, no one seems to have the
complete answer, but something in the range 20-100 uM
cycloheximide would seem to be a good starting point for a short-
term treatment to knockout protein synthesis in Arabidopsis
roots. An appropriate control in our case might be to
find the minimum concentration that blocks the nitrate-induced
accumulation of nitrate reductase protein, as determined by
western blots.
We did CHX (cycloheximide) studies in cell cultures, but not in
intact plants. 20 microM final concentration did work nicely.
Control: Ubiquitin mRNA accumulation. We have not yet done
experiments with plants, but maybe root cultures growing in
liquid medium are a possibility.
Bernd Weisshaar e-mail: weisshaa at mpiz-koeln.mpg.de
I have used cycloheximide in many experiments for the purpose you
describe. I mostly work with tobacco, but I have successfully
used CHX with arabidopsis too. In both cases, however, I
generally work with leaf tissue, so I don't know about how
well CHX gets into roots. In our experiments, we seem to be able
to distinguish translation-independent and translation-dependent
transcription, and have demonstrated reduced translation
efficiency by incorporation of labelled methionine (Qin, et al,
1994, June Plant Cell). I say 'seem' because one cannot
completely rule out a secondary effect, say on protein
phosphorylation cascades as has been argued by
some (I can look up the reference, if you are interested).
Diana Horvath email: ralston at rockvax.rockefeller.edu
(NB - Qin et al. used 20 ug/ml cycloheximide = ca 70 uM)
For what it's worth, I have found that root growth in arabidopsis
is inhibited by cycloheximide. Seven day old seedlings were
transferred to plates with different concentrations and growth
was measured over the next 2d. The threshold concentration was
30 nM, and by 100 nM elongation was about 80% inhibited. Sorry,
I did not measure protein synthesis. Nor did I do any rapid
growth kinetics. There was a paper in last year's Plant
Physiology by Mineyuki et al (1994 Plant Phys 104:281) which
showed protein synthesis in onion root tips was inhibited rapidly
(within an hour?) by cycloheximide.
Tobias Baskin email: baskin at biosci.mbp.missouri.edu
I tried cycloheximide (10 uM for 2 h) on Arabidopsis root
cultures. It resulted in a super-induction of a rapid auxin-
responsive mRNA. I didn't measure protein synthesis, though,
so I can't help you much.
Marta Laskowski email: mlaskow at garnet.berkeley.edu
With regards to your question about protein synthesis inhibitors,
you are probably already aware of the following studies on
maize: G Gowri et al. (1992) Plant Molec. Biol. 18:55-64; MG
Redinbaugh and WH Campbell (1993) Plant Physiol. 101:1249. They
treated seedling roots for 3-4 h in hydroponic solutions with
~50-85 ug/ml CHX (180-300 uM). Undoubtedly, continued treatment
would prove fatal, but for the purposes of demonstrating a
primary response to nitrate, only short-term exposure is
required. Appropriate controls are necessary, of course.
Paul Meyerhoff email: pmeyerhoff at ucdavis.edu